Note: Descriptions are shown in the official language in which they were submitted.
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
RARE EVENT DETECTION SYSTEM
Cross Reference to Related Applications
This application claims benefit ofU.S. Application Serial No. 60!265,909,
entitled
Rare Event Detection System, filed February 2, 2001, which application is
hereby
incorporated by reference in its entirety.
Statement as to Federally Sponsored Research
This invention was made with Government funds through a grant (CA13849) from
the National Institutes of Health. The Government has certain rights in the
invention.
Background of the Invention
Most human cancers are characterized by the aberrant expression of normal
andlor
mutated genes, and natural selection acts on cancer cells to cause a loss of
growth control,
angiogenesis, invasion, and metastasis. Thus, the ability to detect cancer
cells of particular
phenotypes in patient samples provides valuable information to a health care
provider. For
example, if the presence of metastatic cancer cells is detected in the body,
then a medical
professional might consider a more aggressive therapy for the patient.
Cancer cell detection methods that rely on expression of cancer markers
generally
require long, labor-intensive, and sometimes expensive immunohistochemistry or
nucleic
acid hybridization procedures that, though ubiquitous in research
laboratories, are less
accessible in the clinic. Furthermore, in many instances the particular marker
being screened
is only produced, either initially, or in detectable levels only at a late
stage of cancer
progression, such that the advantage of early detection is squandered. Current
technologies
allow detection of micrometastasis along the order of 1 parts-per-million
(i.e., one cancer cell
per one million other cells), however, this detection level is still
inadequate for true "early
detection" in certain cancers. More sensitive levels of detection would
effectively provide
cancer cell detection capabilities to allow appropriate and more effective
intervention of
cancer cell proliferation and thereby more effective and timely cancer
treatment and disease
modulation therapies. Thus, there is a need for fast, efficient, reliable, and
sensitive detection
methods that are more amenable for use in the clinic.
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
The detection of biological weapons (BW) on a battle field poses a similar
problem,
i.e., no suitable method or device for detecting a rare particle (e.g., toxin
or virus) among a
large population of particles. Biological weapons, defined as infectious
agents such as
bacteria and viruses or related toxins, when used intentionally to inflict
harm upon others,
have been with us for a long time. They were probably originally used in
prehistoric times,
as arrowheads dipped into plant or animal extracts containing toxins; or in
fecal matter or
decaying meat, which are sources of the gas gangrene bacterium, Clostridium
perfi~i~gens,
and often also of the tetanus bacillus, C. tetahi. BW first appear on the
record as early as the
6th Century BC when the Assyrians poisoned enemy wells with rye ergot; and
Solon of
Athens used the purgative herb hellebore (skunk cabbage) to poison the water
supply during
the siege of Krissa; the Romans and many others have used a similar strategy;
and during the
14th Century AD, the Mongols are said to have catapulted plague-infected
corpses over the
city walls of Kaffa, which they were besieging, an event that may have started
the Black
Death pandemic that spread throughout Europe. Other examples for the crude or
more
sophisticated use of BW abound, up to the late 20th Century.
Advances in basic and applied microbiology now allow skilled scientists to
harness
and weaponize the most virulent pathogens and toxins. While several countries
(including
the United States) have developed BW programs at some point or another during
the 20th
century, efforts in Japan and in the former Soviet Union are perhaps the most
notorious.
From 1932 until the end of WW II, the Japanese Army engaged in biological
weapons
research through its "Unit 731," based in occupied China. Research with human
subjects
(Chinese and Russian civilians and American, British, Chinese, Korean and
Russian
prisoners of war) was conducted using a variety of agents including anthrax,
glanders,
plague, typhoid, paratyphoid A and B, typhus, smallpox, tularemia, infectious
jaundice, gas
gangrene, tetanus, cholera, dysentery, scarlet fever, undulant fever, tick
encephalitis,
whooping cough, diphtheria, pneumonia, venereal diseases, tuberculosis and
Salmonella.
The Soviet program was initiated in 1928, when the governing Revolutionary
Military
Council signed a secret decree ordering the transformation of typhus into a
battlefield
weapon. In the 1930's, scientists at the Solovetsky Island facility, in the
Arctic, worked with
typhus, Q-fever, glanders, and melioidosis. From 1973 through at least the
early 1990's, the
Soviet Union carried out a program aimed at modernizing existing biological
weapons and at
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
developing genetically altered pathogens, resistant to antibiotics and
vaccines, which could
be turned into powerful weapons for use in intercontinental warfare. Agents
studied included
anthrax, turalemia, plague, glanders, smallpox, Ebola, Marburg, Machupo,
Junin, and
Venezuelan equine encephalitis.
All open societies, such as ours, are by their very nature vulnerable to
terrorist
attacks, both from international and domestic groups. With this state of
affairs, it is most
urgent that effective countermeasures be developed to preempt biological
attacks, or render
them ineffective through the protection of the target population (troops or
civilians).
Biological weapons have a few unique features that make them especially
formidable.
For one, hurdles would be few for a small team comprising a competent
microbiologist and a
mechanical engineer, to grow or extract a variety of pathogenic agents
(bacteria, viruses or
toxins) and build an effective dispersion system: it has been estimated that a
major biological
arsenal could be built in a room 15 by 15 ft., with X10,000 worth of
equipment. This makes
BW tools of choice for groups bent on terrorism who may want to inflict
massive casualties
to their opponent. Also, contagion may in some cases expand the outcome of the
attack well
beyond the confines of the original hit, both geographically and temporally.
Finally, the
actions of BW agents on the victims are generally delayed by at least hours,
usually days,
allowing a covert attack to be sustained during this period (besides giving
the perpetrators an
opportunity to flee, another boon for the stealth terrorist), and the early
symptoms of an
infection with a variety of BW pathogens are flu-like, making it very
difficult to quickly
recognize a BW attack as such.
Our ability to respond effectively to a biological attack on an unimmunized
population therefore depends crucially on the development of new modalities
for the rapid
monitoring of BW agents in the environment, both airborne (indoors and
outdoors) and
waterborne, before an outbreak of the disease. This is also the time window
when early
detection of pathogens in human body fluids, e.g., blood, prior to the
appearance of clinical
symptoms is important.
Theoretically, any pathogen could be used as a biological weapon. However,
certain
characteristics make a biological organism or a biologically derived bioactive
substance
(BDBS), such as bacterial toxins, especially suited for use as weapons of mass
destruction.
These agents can be: 1) highly infectious, contagious, and toxic (i.e., even
low-level
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
exposure causes disease); 2) efficiently dispersible, e.g., in the air; 3)
readily grown and
produced in large quantities; 4) stable in storage; 5) resistant to
environmental conditions, for
extended effect; and 6) resistant to treatment, e.g., antibiotics, antibodies,
other drugs.
To the list of natural pathogens, one should add genetically modified BW
agents.
This class of agents is particularly dreadful because they would be generated
to make them
more potent, even creating new diseases (e.g., resulting in a "brainpox"
virus), or produce
pathogens resistant to existing countermeasures. These pose a special
challenge due to their
unpredictability.
For the reasons described above, a covert attack using BW agents would be
extremely
difficult to detect and assess (in the absence of intelligence). At the
present time, as no
formal environmental monitoring system exists, the earliest knowledge that an
attack took
place would occur in many cases only when victims start pouring into emergency
rooms and
an outbreak is recognized. This, of course, is far too late. The classical
monitoring methods
for pathogens involve environmental sampling (air, water supply) or body fluid
sampling
(blood, urine, sputum etc.) onto growth media and culture of the sample
followed by a
battery of microbiological tests to identify the culprit. In addition to the
fact that culture is
not a trivial endeavor (e.g., for viruses), such a procedure is much too
lengthy to provide a
timely alert. Other possible analysis methods include biochemical assays,
immunoassays,
"GeneChip" screening, and the polymerase chain reaction (PCR), but all these
require
amounts of the contaminant that may not be present in the initial sample (to
meet a sensitivity
commensurate with an actual threat), such that culture may still be needed;
even PCR from a
single bacterium or virion is impractical.
Summary of the Invention
The invention is based on technologies that provide for detecting the presence
of a
rare event or marker. The invention relates to equipment and methods for
identifying,
characterizing (either quantitatively, qualitatively, or both), analyzing or
determining the
presence of minute quantities of rare events or markers. The determination of
the presence or
absence of such rare events or markers, as well as the quantification of such
rare events or
markers, is useful in providing early detection of deleterious or potentially
harmful entities or
conditions, which if identified earlier rather than later, can allow for the
application of an
4
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
appropriate response, treatment, or other intervention regimen or protocol.
Rare events
include both normal events (e.g., the presence or absence of target bodies or
cells that are
present in normal physiological states) and abnormal events (e.g., the
presence or absence of
target bodies or cells that are present in abnormal physiological states such
as those
associated with disease, disease symptoms, or genetic abnormalities). One
problem with
current diagnostic methods, particularly for cancer, relates to minimal
residual disease. That
is, instances when the level of disease cells or other disease markers (e.g.,
nucleic acids,
proteins, cell surface receptors) is too low for current detection methods,
however, significant
enough that they represent the potential for further proliferation, up-
regulation or recurrence
of the disease if left undiagnosed or untreated. Thus, in many instances,
identification of
disease risk (i.e., cancer, artherosclerosis, central nervous system disease,
etc.) in a more
timely manner would allow for earlier treatment, which leads to more effective
treatments; or
earlier identification of risk to populations (i.e., biological warfare
agents), which allows for
minimization of exposure and uncontrolled spreading or distribution of that
risk to greater
populations, is desirable.
The invention is based on the discovery of a highly sensitive and efficient
method of
detecting rare cancer cells in a large cell population. In addition, the
cancer cell detection
system implemented herein led to the realization that almost any rare target
body within a
large population of candidate bodies can be detected via this system, modified
for the
particular target body to be identified. The methods and systems of the
invention rely on
fluorescent labels that specifically bind to subsets of a large population,
each subset including
the target body to be detected. A target body is any body (e.g., a cell, a
pathogen, a virus, a
toxin, a prion) in the specimen field that is sought to be identified (e.g.,
by labeling, including
directly to the target body or indirectly such as when the label is coupled to
an molecule that
binds or interacts with the target body). A candidate body is any body (e.g.,
a cell, a
pathogen, a virus, a toxin, a prion) in the specimen field that is being
analyzed.
Accordingly, the invention features a method of detecting a target body (e.g.,
a cancer
cell) in a specimen by obtaining a specimen field (e.g., peripheral blood
mononuclear cells
(PBMC) or bone marrow cells spread out on a glass surface) exposed to or
labeled with at
least a first fluorophore and a second fluorophore, the first fluorophore
emitting photons at a
first wavelength and the second fluorophore emitting photons at a second
wavelength;
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
exposing the specimen field to light sufficient to excite the first and second
fluorophores;
scanning the specimen field for first sources of photons at the first
wavelength and for second
sources of photons at the second wavelength; acquiring and recording a first
image of the
specimen field at each location, the first image generated via an optical or
electronic filter
that substantially blocks photons of the second wavelength but is permissive
for photons of
the first wavelength and; indexing the corresponding location within the
specimen field;
acquiring and recording a second image of the specimen field at each location,
the second
image generated via an optical or electronic filter that substantially blocks
photons of the first
wavelength but is permissive for photons of the second wavelength; indexing
the
corresponding location within the specimen field; and retrieving and
inspecting a first image
and second image at a single location within the specimen field. The presence
of a candidate
body in the first and second images at the single location indicates the
presence of a target
body in the specimen. Images of different fluorescent signals can be overlaid
for positive
confirmation of the event or for phenotypic evaluation. The two scans can be
run
independently.
The first fluorophore can be a compound that specifically binds to DNA, such
as
DAPI, or RNA, such as acridine orange. The second fluorophore can be coupled
to a
molecule (e.g., an antibody or nucleic acid) that specifically binds to a
cancer cell marker,
such as cytokeratin or another marker.
In some embodiments, the specimen field can be labeled with a third
fluorophore to
increase the specificity of the rare event detection or to detect multiple
subsets of target
bodies, for example a cancer cell and a virus, and the method can further
include exposing
the specimen field to light sufficient to excite the third fluorophore, the
third fluorophore
emitting light at a third wavelength; scanning the specimen field for third
sources of photons
at the third wavelength; registering the location of each third source within
the specimen
field; acquiring and recording a third image of the specimen field at each
location, the third
image generated via an optical or electronic filter that substantially blocks
photons of the first
and second wavelength but is permissive for photons of the third wavelength;
indexing each
third image to the corresponding location within the specimen field; and
retrieving and
inspecting a third image at the single location within the specimen field. The
presence of a
candidate body in the first, second, and third images at the single location
indicates the
6
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
presence of a target body. The third fluorophore can be coupled to a molecule
(e.g., an
antibody) that specifically binds to a second cancer cell marker such as an
epithelial cell
adhesion molecule (e.g., Ep-CA1V~ or a disialo-ganglioside antigen (e.g.,
GD2).
The methods can further include counting the total number of locations in the
specimen field that produced a first image, counting the total number of
locations in the
specimen field that produced both a first image and a second image, or
counting the total
number of locations in the specimen field that produced a first, second, and
third image. In
addition, the methods can include inspecting a first image and second image at
another single
location within the specimen field, where the presence of a candidate body in
the first image
and in the second image at the other single location indicates the present of
a different target
body.
The invention further features a detection system including a stage for
receiving a
specimen field; a detector (e.g., microscope) positioned and configured to
acquire images of
locations within the specimen field; a light source positioned and configured
to expose the
specimen field to light sufficient to excite a first fluorophore at a first
excitation wavelength
and sufficient to excite a second fluorophore at a second excitation
wavelength; a camera
attached to the detector (e.g., microscope), the camera positioned and
configured to (1)
capture a first image at a location in the specimen field via an optical or
electronic filter that
substantially blocks photons at a second emission wavelength of the second
fluorophore but
is permissive for photons at a first emission wavelength of the first
fluorophore, and (2)
capture a second image at the location in the specimen field via an optical or
electronic filter
that substantially blocks photons at the first emission wavelength but is
permissive for
photons at the second emission wavelength; and a computer that records the
first image and
second image and indexes the first image and second image to the corresponding
location
within the specimen field, the computer displaying, on demand by a user, the
first image and
second image for the corresponding location.
The stage can be movable about three perpendicular axes and addressable in at
least
two of the three axes. Alternatively, the camera or a housing containing the
camera andlor
image capture device can be movable about three perpendicular axes and
addressable in at
least two of the three axes. The camera can include a charge-coupled device
for capturing
the first and second images or a plurality of optical filters for use in
capturing the first and
7
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
second images. Alternatively or in conjunction with optical filters, the
cameral or computer
can include electronic filters. Such filters can dissect a digitized color
image taken at a range
of wavelengths (e.g., the visible wavelengths) into images formed at only
specific
wavelengths or narrower ranges of wavelengths.
In another aspect, the invention features a method of detecting a target body
in a
specimen by obtaining a specimen field labeled with at least a first
fluorophore, the first
fluorophore emitting photons at a first wavelength; exposing the specimen
field to light
sufficient to excite the first fluorophore; scanning the specimen field at a
low magnification
for first sources of photons at the first wavelength; acquiring and recording
a first image of
the specimen field at each location; indexing each first image to the
corresponding location
within the specimen field; and inspecting a first image at a single location
within the
specimen field, where the presence of a candidate body in the first image at
the single
location indicates the presence of a target body in the specimen.
The methods and systems of the invention are capable of fast, highly
sensitive, and
efficient detection of rare target bodies within a large population of
candidate bodies, such as
a rare cancer cell within a million healthy cells, a level of sensitivity
achievable with the
present invention. The methods and systems herein allow for detection levels
along the order
of about 0.1 parts-per-million, or commensurately more beneficial, about 0.05,
about 0.03, or
about 0.01 parts-per-million.
In one aspect the invention is a method of detecting the presence or absence
of a
target body in a specimen, the method comprising
obtaining a specimen field exposed to or labeled with at least a first
fluorophore and a second fluorophore, the first fluorophore emitting photons
at a first
wavelength and the second fluorophore emitting photons at a second wavelength;
exposing the specimen field to light sufficient to excite the first and second
fluorophores;
scanning the specimen field at a low magnification for first sources of
photons
at the first wavelength and for second sources of photons at the second
wavelength;
registering the location of each first source and each second source within
the
specimen field;
8
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
acquiring and recording a first image of the specimen field at each location,
the first image generated via an optical or electronic filter that
substantially blocks photons of
the second wavelength but is permissive for photons of the first wavelength;
acquiring and recording a second image of the specimen field at each location
at a high magnification, the second image generated via an optical or
electronic filter that
substantially blocks photons of the first wavelength but is permissive for
photons of the
second wavelength;
indexing each first image and each second image to the corresponding
location within the specimen field; and
inspecting a first image and second image at a single location within the
specimen field,
wherein the presence of a candidate body in the first and second images at the
single location indicates the presence of a target body in the specimen.
In another aspect the invention is any method herein wherein preparation of
the
specimen field comprises:
a. lysing the cell sample to give a sample mixture;
b. centrifuging the sample mixture;
c. separating the supernatant from the sample mixture;
d., resuspending the resulting pellet of cells in a physiological buffer
solution;
e. plating the cells on an adhesive slide;
f. adding cell culture media to the slide.
and wherein preparation of the specimen field further comprises:
after step d, making a dilution of the cell mixture, treating the dilution
with a dye
sensitive for dead cells, performing a cell count to determine the sample cell
density for the
slide to be used.
In other aspects, the methods are any of those herein: wherein the target body
is a
cancer, epithelial, smooth muscle, dendritic, memory T , memory B-, somatic,
normal,
aberrant, or stem cell; wherein the system is capable of detecting at least
one target cell in a
specimen field of at least 1,000,000 cells; wherein the system is capable of
detecting at least
one target cell in a specimen field of at least 25,000,000 cells; wherein the
system is capable
of detecting at least one target cell in a specimen field of at least
50,000,000 cells; wherein
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
the system is capable of detecting at least one target cell in a specimen
field of at least
100,000,000 cells; wherein the recording comprises at least a 1024x1024 pixel
array image;
or wherein the recording comprises at least a 1600x1600 pixel array image.
In other aspects, the methods are any of those herein: wherein the field
specimen
comprises white blood cells as the majority of cell types; wherein the field
specimen
comprises heterogeneous cells types; wherein the field specimen comprises
macrophages;
wherein the specimen field is an environmental sample; wherein the light is
ultraviolet light,
infrared light, or visible light; wherein the target body is a cancer cell,
and the specimen field
is white blood cells or bone marrow cells spread out on a glass surface;
wherein the first
fluorophore is a compound that specifically binds to DNA; wherein the second
fluorophore is
coupled to a molecule that specifically binds to a cancer cell marker; wherein
the cancer cell
marker is cytokeratin; wherein the cancer cell marker resides in the
cytoplasm; wherein the
cancer cell surface marker is an epithelial cell adhesion molecule; wherein
the cancer cell
surface marker is a disialo-ganglioside antigen; further comprising counting
the total number
of locations in the specimen field that produced a first image; further
comprising counting the
total number of locations in the specimen field that produced both a first
image and a second
image; further comprising counting the total number of locations in the
specimen field that
produced a first, second, and third image; further comprising inspecting a
first image and
second image at another single location within the specimen field, wherein the
presence of a
candidate body in the first image and in the second image at the other single
location
indicates the present of another target body.
In another aspect , the invention is a detection system comprising
a stage for receiving a specimen field;
a detector positioned and configured to acquire images of locations within the
specimen field at a set level and one or more additional amplifications of the
set level;
a light source positioned and configured to expose the specimen field to light
sufl cient to excite a first fluorophore at a first excitation wavelength and
sufficient to excite
a second fluorophore at a second excitation wavelength;
a camera attached to the detector, the camera positioned and configured to (1)
capture a first image at a location in the specimen field via an optical or
electronic filter that
substantially blocks photons at a second emission wavelength of the second
fluorophore but
to
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
is permissive for photons at a first emission wavelength of the first
fluorophore, and (2)
capture a second image at the location in the specimen field via an optical or
electronic filter
that substantially blocks photons at the first emission wavelength but is
permissive for
photons at the second emission wavelength; and
a computer that records the first image and second image and indexes the first
image and second image to the corresponding location within the specimen
field, the
computer displaying, on demand by a user, the first image and second image for
the
corresponding location.
In other aspects, the system is any herein wherein the stage is movable about
three
perpendicular axes and addressable in at least two of the three axes; wherein
the camera
comprises a charge-coupled device for capturing the first and second images;
wherein the
camera comprises a plurality of optical filters; wherein the detector
comprises a 1024x1024
pixel array image; wherein the detector comprises a 1600x1600 pixel array
image; or
wherein the detector comprises an A x B pixel array image, wherein A and B are
each
independently an integer between, 1000 and 1,000,000.
The invention also relates to a method for analyzing for biological agent
cells in a
specimen field of cells comprising:
i) treating the specimen field with a first fluorophore that identifies the
biological agent cell;
ii) treating the specimen field with a second fluorophore that identifies
the biological agent cell;
iii) exposing the specimen field with light suitable for causing the first
fluorophore to emit photons,
iv) exposing the specimen field with light suitable for causing the second
fluorophore to emit photons,
v) identifying cells in the specimen field that are emitting photons, which
cells are biological agent cells.
In another aspect, the invention is any method herein: wherein the specimen
field cell
preparation comprises:
a. centrifuging a sample mixture;
b. resuspending the sample mixture;
11
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
c. plating the cells on an adhesive slide;
d. treating the slide with a fixative (paraformaldehyde);
e. treating the slide with a permeabilizing agent (Triton);
f. treating the slide with a pre-hybridization solution;
g. treating the slide with a hybridization solution having a fluorophore;
h. treating the slide with a fluorescent dye.
and that further comprising treating the specimen field with one or more
additional
fluorophore(s) that identifies the biological agent cell and exposing the
specimen field with
light suitable for causing the one or more additional fluorophore(s) to emit
photons.
In other aspects, the invention relates to any method herein: wherein at least
one
fluorophore identifies DNA of a biological agent cell; wherein at least one
fluorophore
identifies a molecule that binds to the surface of the biological agent cell;
wherein at least
one fluorophore identifies DNA of a biological agent cell and at least one
fluorophore
identifies a molecule that binds to the surface biological agent cell; or
wherein the biological
agent is bacteria, Rickettsiae, viruses, fungi, or prions.
In another aspect, the~invention is any method herein: wherein preparation of
the
specimen field comprises:
a. lysing the blood sample with ammonium chloride solution;
b. centrifuging the sample mixture;
c. separating the supernatant ammonium chloride solution and erythrocytes;
d. resuspending the resulting pellet of white cells in PBS;
e. centrifuging the sample mixture;
f. resuspending the resulting pellet of white cells in PBS;
g. making a dilution of the cell mixture of step f, tryphan blue, and PBS;
h. plating the cells on an adhesive slide;
i. adding cell culture media to the slide.
and that further wherein one fluorophore identifies cells that are not target
cells. In other
aspects the methods are those wherein the method is completed for a specimen
field in less
than 60 minutes; or wherein the method is completed for a specimen field in
less than 10
minutes.
12
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
In other aspects, the invention is a method for screening a transplantation
organ donor
for the presence or absence of a target body comprising any method herein,
wherein the
specimen field is a sample (e.g., blood sample, tissue sample) taken from the
organ donor.
This is useful for identifying target bodies in the donor prior to
transplantation, thus
preventing spread of those bodies to the donee. The invention also relates to
a method for
assessing the efficacy of a drug candidate against a disease or disease
symptom in a subject
who was administered the drug candidate by screening for the presence or
absence of a target
body whose presence or absence is indicative of the disease or disease symptom
comprising
any method herein, wherein the specimen field is a sample taken from the
subject. The
invention also relates to a method for screening a blood sample for the
presence or absence of
a target body comprising any method herein, wherein the specimen field is a
blood sample.
This is useful for identifying contaminated blood samples, for example in
blood banks, prior
to distribution of those contaminated samples. It could also be used for
screening potential
donors prior to their donation. The invention is also a method for screening a
fluid sample
for the presence or absence of a target body comprising any method herein,
wherein the
specimen field is a fluid sample; and any method herein, wherein the target
body is a cancer
cell.
In another aspect, the invention is a method of screening for the presence of
bacteria
comprising any method herein: wherein at least one fluorophore comprises a DNA
probe for
bacteria; wherein the specimen field is taken from a surgical patient after
surgery; wherein
the specimen field is taken from a food sample; or any method herein further
comprising:
j. exposing the slide to an aldehyde-based fixative;
k. rising the slide in phosphate-buffered saline (PBS);
1. adding human AB serum to the slide;
m. adding a primary antibody to the slide and incubating the slide;
n. rinsing the slide in PBS;
o. adding a secondary antibody to the slide and incubating the slide;
p. exposing the slide in an organic solvent;
q. rinsing the slide in PBS;
r. adding human AB serum to the slide;
s. adding a primary antibody to the slide and incubating the slide;
13
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
t. rinsing the slide in PBS;
u. adding a secondary antibody to the slide and incubating the slide;
v. rinsing the slide in PBS;
w. adding a cell dye to the slide and incubating the slide;
x. rinsing the slide with PBS;
y. exposing the slide to water;
z. mounting the slide;
or wherein the primary antibody in step s is keratin and the secondary
antibody in step a is
anti-rabbit rhodamine;
or any method herein further comprising:
j. exposing the slide in an organic solvent;
k. rinsing the slide in PBS;
1. adding a primary antibody to the slide and incubating the slide;
m. rinsing the slide in PBS;
n. adding a secondary antibody to the slide and incubating the slide;
o. rinsing the slide in PBS;
p. adding a cell dye to the slide and incubating the slide;
q. rinsing the slide with PBS;
r. exposing the slide to water;
s. mounting the slide;
or those: wherein the organic solvent is an alcohol or acetone; wherein the
primary antibod;
is keratin; wherein the secondary antibody is anti-rabbit rhodamine; wherein
the fluoropho~
detects bacteria; wherein the fluorophore is a nucleic acid probe; or wherein
the nucleic aci
probe is an oligonucleotide.
Other features or advantages of the present invention will be apparent from
the
following detailed description, and also from the claims.
Detailed Description
The invention relates to fluorescence-based methods and systems for detecting
rare
target bodies within a large number of candidate bodies. Because a wide
variety of
fluorophores are commercially available and have different peak emission
wavelengths, thf
14
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
methods and systems can be adapted to detect many different target bodies
within a single
large population of candidate bodies. For example, fluorophores A, B, C, D, E,
and F can b
coupled to molecules that specifically bind to target bodies 1, 2, 3, 4, 5,
and 6, respectively.
One merely needs to capture and assess the emission wavelength, if any, of a
candidate bod:
S and compare the emission wavelength with what would be expected from
fluorophores A-F
to determine whether the candidate body is a target body 1, 2, 3, 4, 5, or 6.
In fact, far large
numbers of targets can be detected simultaneously in this manner. Additional
details
regarding the various reagents and procedures suitable for use in the
invention are discusses
below.
Preparation of Specimens for Detection
In common clinical applications, a specimen will typically be a cell sample in
body
fluids, bone marrow, or a tissue sample, e.g., a blood cell sample, that can
be screened for
the presence of a rare cell having a particular phenotype (using, e.g.,
antibodies) or genotyp
(e.g., using oligonucleotide probes).
The cell specimen preparation methods herein result in enrichment for cell
types
desired for analysis. This can be accomplished by any suitable method for
separating or
isolating cells, including for example, gradient separation, or lysis and
centrifugation.
For the automated detection of rare events in peripheral blood or bone marrow,
it is
important to utilize a preparation method with minimal cell loss during sample
processing.
Simple lysis of erythrocytes (e.g., using ammonium chloride solution) is
preferred over
Ficoll-based isolation methods to ensure maximal recovery of rare cells.
Performing the 1y;
in the same tube containing the blood sample, then performing the separation
(e.g.,
centrifuging, spinning down) in the same tube (i.e., involving no transfer of
sample during
the lysis and separation) also minimizes cell loss and minimizes cell
representation variation
in the sample (i.e., maintaining a consistent relative proportion of rare
cells to other cells in
the sample both before and after processing). The cell preparation/adhesion
procedure
described in the Example below yielded a homogeneous cell preparation.
In contrast, regular cytospin preparations can result in a loss of up to 2/3
of the cells
Information on cell number is unavailable for most studies using microscopic
rare event
detection because these studies fail to record the total number of cells
actually being analyze
on the slides. Rather, these experiments merely relate the number of positive
events to the
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
total number of cells processed, assuming a complete recovery. This introduces
a bias: not
only was it found that cells are indeed inevitably lost during preparation,
but the recovery cap
vary greatly between samples of a given type (see "Range" column in Table 1)
as well as
according to the type of sample analyzed. It was found that adhesive glass
microscope slides
from Marienfeld Laboratory Glassware (Paul Marienfeld GmbH & Co;
www.superior.de)
were excellent substrates for producing a cellular specimen field for
subsequent fluorescence
microscopy, because these slides were able to capture a homogenous cell
monolayer (optima
cell density with minimal overlap). Once the media is introduced to the slide,
treatment with
any aldehyde-based fixative (e.g., paraformaldehyde, formalin, gluteraldehyde,
cross-linking
agent) fixes the cells. In certain cells types where the antigen is not at the
cell surface, the
cells can be permeablized, using a permeablizing agent (e.g., methanol,
TRITON). If the
antigen is a surface antigen, then the permeablization is not required.
Exposure of the slides
to an organic solvent (e.g., alcohols, ketones, methanol, ethanol, acetone)
can be used to
permeablize the cells, and certain solvents (e.g., methanol) can both fix and
permeablize.
Cell culture media can be any media that can cover free binding sites, or can
have proteins,
including for example RPMI or DMEM. Physiological buffer solutions are those
that are
compatible with cells and include for example, any isotonic solution, or PBS.
Cell dyes are
any dye suitable to stain a cell and include for example, DNA dyes,
cytoplasmic dyes,
mitochondria) dyes, DAPI, calcein and the like. With the proper specimen
preparation, any
unexpected cell type in a biological tissue or fluid can be detected using the
invention. For
example, the presence of smooth muscle cells in blood may indicate
atherosclerosis. In
another example, packaged blood in a blood bank can be screened for the
existence of
common pathogens transmitted by transfusion, such as human immunodeficiency
virus,
hepatitis B virus, or cytomegalovirus.
Whatever method is used to prepare the specimen field for analysis, it is
important
that the method does not destroy or significantly alter the target body to be
detected. For
example, if the target body is a prion, bacteria, virus, protozoan, or
multicellular parasite, th
isolation procedures may differ. Analysis of solid tissue (e.g., a solid
tumor) may require
disaggregating cells, e.g., by physical disruption instead of by
trypsinization, since protease
treatment can alter any cell surface molecule that is used to identify a
target cell. Preparatio
of a virus specimen field may entail filtering out large particles of a
certain size (e.g., cells)
16
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
so that only sub-cellular particles are present in the specimen field.
Alternatively, cells can
be included in the specimen field if detection of virus-infected cells is
desired. Various well
known preparation procedures for particular biological samples are available
to one skilled in
the art of pathology and microscopy, and these procedures can be adapted to
whatever target
bodies are to be detected. Such procedures include cytospin using a Shandon
Cytocentrifixge,
Cytotek Monoprep from Sakura (Torrance, CA), and ThinPrep from Cysyc
(Boxborough,
MA).
When the sample to be analyzed is not a biological fluid such as blood,
different
devices can be used to collect samples from, e.g., air. In general, an air
sampling device has
a collection chamber containing liquid through or beside which air or gas is
passed through,
or containing a porous filter that traps particulates (e.g., target bodies) as
air or gas passes
through the filter. For collection chambers containing liquid, the collection
liquid can be
centrifuged or otherwise treated to separate particles from the liquid. The
separated particles
are then deposited onto a substrate for labeling or analysis. For collection
chambers
containing a filter (e.g., nitrocellulose), the filter can act as a substrate
for subsequent
labeling or analysis. Alternatively, particles can be washed from the filter,
or the filter can be
dissolved or otherwise removed from the particles. A filter collection chamber
can also be
adapted to collect particles from a liquid (e.g., water supply sample or
cerebral spinal fluid)
flowing through the filter. In addition, as discussed above, a liquid sample
can be centrifuged
to remove any particulate material present in the liquid. In instances when
the test material
remains in solution in the liquid sample and undesirable particulate matter is
removed (e.g.,
by filtration), the mother liquor can be sampled (either in solution, or upon
ira vacuo drying of
the sample solution) for analysis. A variety of samplers are known and
available for use
with the present invention. See SKC, Inc. (www skc.com), which sells the SKC
BioSampler and other sampling devices.
Tt is contemplated that the invention encompasses detection of biological
warfare
agents or any agent that is harmful to humans, animals, or plants. In that
light, the methods
and systems of the invention can be used to detect agents harmful to humans,
commercially
valuable animals, or commercially valuable plants. Human bacteria and
Rickettsiae agents
include but are not limited to Coxiella burnetii, Bartonella Quintarra
(Rochalimea quintana,
Rickettsia quintana), Rickettsia prowasecki, Rickettsia rickettsii, Bacillus
anthraci, Brucella
17
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
abortus, Brucella melitensis, Brucella suis, Chlarnydia psittaci, Clostridium
botulir~um,
Frarrcisella tularensis, Burkholderia mallei (Pseudomorras mallei),
Burkholderia
pseudomallei (Pseudomonas pseudomallei), Salmonella typhi, Shigella
dysenteriae, brio
cholerae, Yersinia pesos, Clostridium perfringens, Cl~stridium tetani,
Enterohaemorrhagic
Escherichia coli (serotype 0157 and other verotoxin producing serotypes),
Legionella
pneumophila, and Yersinia pseudotuberculosis. Human viral agents include but
are not
limited to Chikungunya virus, Congo-Crimean hemorrhagic fever virus, Dengue
fever virus
Eastern equine encephalitis virus, Ebola virus, Hantaan virus, Junin virus,
Lassa fever virus
Lymphocytic choriomeningitis virus, Machupo virus, Marburg virus, Monkey pox
virus, R.i
Valley fever virus, Tick-borne encephalitis virus, Variola virus, Venezuelan
equine
encephalitis virus, Western equine encephalitis virus, White pox, Yellow fever
virus,
Japanese encephalitis virus, Kyasanur Forest virus, Louping ill virus, Murray
Valley
encephalitis virus, Omsk hemorrhagic fever virus, Oropouche virus, Powassan
virus, Rocio
virus, and St. Louis encephalitis virus.
Animal bacteria and Rickettsiae agents include but are not limited to
Mycoplasma
mycoides and Bacillus anthracis. Animal viral agents include but are not
limited to African
swine fever virus, Avian influenza virus 2, Bluetongue virus, Foot and mouth
disease virus,
Goat pox virus, Herpes virus (Aujeszky's disease), Hog cholera virus (Swine
fever virus),
Lyssa virus, Newcastle disease virus, Peste des petits ruminants virus,
Porcine enterovirus
type 9 (swine vesicular disease virus), Rinderpest virus, Sheep pox virus,
Teschen disease
virus, and Vesicular stomatitis virus.
Plant bacteria and Rickettsiae agents including but not limited to Xanthomohas
albilir~earzs, Xanthomonas campestris pv. Citri, Xarathomonas ca»zpestris pv.
Oryzae, and
Xylella fastidiosa. Plant viral agents including but not limited to banana
bunchy top virus.
Prions are correlated with diseases including but not limited to bovine
spongiform
encephalopathies, scrapi, and Creutzfeldt-Jakob disease.
In a particular example, a sample can be prepared as follows. Optimized
preparatio
procedure for the immunocytochemical detection of microorganisms can be
applied to
environmental (air and water) and human (blood and other body fluids) samples.
A
BioSampler~ from SKC, Inc. is used to collect an air sample. The BioSampler~
is a vacuum
driven all-glass impinger device that passes air, via nozzles, tangential to
the surface of the
18
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
collection fluid rather than bubbling air through the fluid. This design
minimizes particle
bounce and reduces re-aerosolization. When operated at an air flow rate of
12.5 Llmin with
water or a liquid of similar viscosity as the collection fluid, the collection
efficiency of the
BioSampler is close to 100% for particles as little as 1 p.m in diameter,
still approximately
90% at 0.5 p.m, and 80% at 0.3 pm. As such, the BioSampler is an excellent
device for the
collection of airborne bacteria, fungi, pollen, and viruses, since most
bacteria are between 1
and 10 ~m in diameter and many viruses have a size in the lower end of this
range (e.g.
Ebola virus, 1000 x 80 nm).
Other air samplers can be used. For example, an alternative device is the Air-
O-Cell
sampling cassette (SKC, Inc.). In this device, the airborne particles are
accelerated and made
to collide with a tacky slide which is directly suitable for various staining
procedures and
microscopic examination. However, this collection method is inei~icient for
particles smaller
than 2 or 3 pm.
The main parameters to be modified in environmental sampling are the time of
sampling and the collection fluid composition. Various fluids can be tested
and compared in
direct inoculation tests with known amounts of organisms, for their capacity
to support
adhesion to the slides.
The analysis of human body fluids are exemplified by the analysis of blood
samples,
as described in Example 1 below.
Fluorescent Staining
An advantage of the present invention is that the invention can be implemented
using
a large library of well known and publically available fluorescent molecules.
Sources
include, for example, Molecular Probes (Eugene, OR), Jackson Immuno Research
(West
Grove, PA), Sigma (St. Louis, MO). These molecules are themselves capable of
specifically
binding to a portion of a target body (e.g., fluorescent DNA dyes), or can be
coupled to
antibodies or nucleic acids that specifically bind to portions of a target
body. See, for
example, Fluorescent and Luminescent Probes for Biological Activity, Ed. WT
Mason,
Academic Press, London, 1993 and Handbook of Fluorescent Probes and Research
Chemicals by RP Haugland, Ed. MTZ Spence, Molecular Probes, 1996. In general,
when
antibodies are used in immunofluorescence, the fluorescent dye is chemically
attached to a
secondary antibody that binds to a primary antibody that is specific for an
antigen on the
19
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
target body or attached directly to a primary antibody. Primary antibodies are
available for a
wide variety of antigens. For example, if the target body is a prion, a prion-
specific antibody
can be used to detect prions in a patient's cerebral spinal fluid to diagnose
Creutzfeldt-Jakob
disease. Primary antibodies suitable for use include anti-GD2 and anti-GD-3
antibodies
(Matreya Inc., Pleasant Gap, PA), anti-HER-2neu antibodies (Dako, Carpinteria,
CA), anti-
KSA/EpCAM antibodies (Dako) and anti-cytokeratin antibodies (Sigma, St. Louis,
MO).
Secondary antibodies suitable for use include those available from Molecular
Probes
(Eugene, OR) and Jackson Immuno Research (West Grove, PA). Between antibody
introduction steps in the slide preparation, PBS washes should be performed.
If the antibody
introduction, however, is a serum blocking reagent, that is, where the
antibodies are
introduced to block nonspecific binding sites in the sample, then a PBS wash
is unnecessary
or even undesirable.
The presence of so many different fluorophores, many of which have different
peak
excitation or emission wavelengths, enables multiplex detection of a large
number (e.g., 24 or
more) of target bodies within a specimen field. In this embodiment, each
antibody can be
specific for only one target body. In addition, multiplexing enables detection
of nested
groups of target bodies to provide greater detection accuracy (e.g., to
minimize false
positives). In the Example below, the DNA stain DAPI was used to identify
target bodies
that were nucleated cells, which can indicate total cell count in a sample and
help confirm
that a fluorescing marker is in fact associated with a cell, as opposed to a
fragment or debris.
Anti-cytokeratin antibodies were then used to identify candidate cancer cell
targets within the
target group of DAPI-positive cells. And finally, antibodies against surface
cancer cell
markers were used to identify and count the subgroup of true cancer cells that
were DAPI-,
cytokeratin, and cell surface antigen-positive. This nesting of fluorescence
staining virtually
eliminated false positive results. Other considerations are described below.
The first requirement for immunocytochemical assays is the generation of
antibodies.
When available commercially or otherwise, existing antibodies directed against
surface or
intracellular target antigens can be acquired. In other cases, the antibodies
must be generated
de novo. Irradiated (killed) samples of the organisms of interest can be
obtained (e.g.,
pathogens from the CDC, USAMRIID, etc.) and provided to, e.g., A&G
Pharmaceutical, Inc.
(Baltimore, MD) for the production of monoclonal antibodies (mAbs) to exposed
epitopes.
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
This company has developed a method for mAb production that provides for rapid
development of hybridomas (< 60 days) at a reasonable cost. If any of these
organisms carry
common surface epitope that would cause cross reaction, or if reliably
"killed" organisms
cannot be obtained, one or several antigens specific to the species can be
obtained. In some
situations, the target body to be detected is a class of targets and not an
individual species
within the class. Thus, an antibody that is class-specific rather than species-
specific would
be desirable. Antigens can be purified, expressed from their cloned genes, or
mimicked by a
chemically synthesized peptide. Antibodies can be directly conjugated with
fluorescent
molecules or used in combination with secondary fluorescently labeled
antibodies. Directly
labeled antibodies can be tested by FAGS analysis for specificity against
other
phylogenetically related species.
The specificity of the detection of cancer~cells in blood or bone marrow
preparations
is typically only as good as the marker and antibodies used in the procedure.
The most
widely used marker is cytokeratin, a cytoskeletal component of epithelial and
carcinoma-
derived cells. Although it has been validated as a valuable marker for breast,
prostate,
gastric, and colorectal cancer in a large number of clinical studies,
cytokeratin is not a true
tumor cell-specific marker and can stain epidermal cells, phagocytic cells
that contain
cytokeratin debris, or dye particles. In such cases, accurate microscopic
confirmation of the
malignant cytology of the immunostained cells is important. Another source of
false-positi~
events is cross-reactive staining of the epithelial or cancer cell marker with
blood or bone
marrow cells, e.g. mucin-like epithelial membrane markers are able to cross-
react with
hematopoietic cells. Indeed, it was found that cytokeratin antibodies can
label PBMC from
healthy blood donors (Table 4 in Example 1). About 17% of the peripheral blood
samples
from normal blood donors exhibited cytokeratin positivity, albeit at a low
level (mean was
1.18 CK+1106 cells). It is not clear whether these CK+ cells in "normal"
samples represent
benign epithelial cells, cross-reacting hematopoietic cells, or cancer cells
disseminated from
an undiagnosed primary carcinoma.
To improve the specificity of cancer cell detection, a double-labeling
protocol was
developed for the simultaneous detection of cytokeratin and epithelial surface
markers, Ep-
CAM and GD2. This procedure dramatically reduced false positives, with only
one doubly
labeled cell among the 77 samples tested (CK/Ep-CAM and CKlGD2; Table 5 in the
21
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
Example), suggesting that the few CK+ cells detected in normal samples were
not of cancer
origin. In addition to the mere detection of cancer cells in blood or bone
marrow samples,
efforts have been made to further characterize the phenotype of rare tumor
cells, e.g. with
respect to their aggressiveness, cell cycle stage, or growth behavior
(Allgayer et al., J.
Histochem. Cytochem. 45:203-212, 1997; Allgayer et al., Cancer Res. 57:1394-
1399, 1997;
Pantel et al., J. Natl. Cancer Inst. 85:1419-1424, 1993; and Riesenberg et
al., Histochem.
99:61-66, 1993). Protocols for multiple marker analysis, combining cytokeratin
labeling
with growth factor receptors or proliferation-associated antigens to analyze
breast cancer
samples (Pantel et al., supra), or combining cytokeratin labeling with
prostate specific
antigen to analyze prostate carcinoma (Riesenberg et al, supra) have been
developed. Also,
in gastric cancer patients, cells that were doubly positive for cytokeratin
and the urokinase
plasminogen activator receptor correlated with high metastatic potential
(Allgayer et al.,
Cancer Res. 57:1394-1399, 1997). A variety of possible additional (cancer-
specific) markers
have been described, e.g. glycoproteins (Franklin et al., Breast Cancer Res.
Treat 41:1-13,
1996), gangliosides (Moss et al., N. Engl. J. Med. 324:219-226, 1991), cell
adhesion
molecules (Ross et al., Exp. Hematol. 23:1478-1483, 1995; and Ross et al.,
Bone Marrow
Transplant. 15:929-933, 1995), and other molecules (Vrendenburgh et al., J.
Hematother.
5:57-62, 1996). The sensitivity, quality, and specificity of the cancer cell
detection method
may improve as new markers become available.
Primary antibodies are available for a wide variety of antigens. For example,
if the
target body is a prion, a prion-specific antibody can be used to detect prions
in a patient's
cerebral spinal fluid to diagnose Creutzfeldt-Jakob disease.
Fluorescently labeled nucleic acids can be used as target body-specific probes
instead
of antibodies. Indeed, there are several reasons why detection using nucleic
acid probes in an
in situ hybridization (ISH) may be desirable: (1) Nucleic acid (NA) probes are
easier,
quicker, and cheaper to generate than antibodies (Abs); (2) NA probes can be
grown at will
and inexpensively (monoclonal Abs too, but not polyclonal); (3) NA probes are
expected to
be more consistent than Abs (especially polyclonal; can even choose probes
with matching
Tm, for multiple labeling (multiplex) experiments); (4) NA probe hybridization
to its cognate
RNA or DNA target can be better controlled than antibody interaction with its
epitope (e.g.,
by hybridization temperature, ionic strength, etc.); (5) Multiple-label
experiments are easier
22
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
to implement with NA probes (simply incorporate a nucleotide conjugated to
different labels,
or incorporate biotin and then various streptavidin-label complexes; in
immunofluorescence
(IF), labeling of primary Ab may interfere with its binding, and when a second
Ab is used for
detection, IF requires the use of primary Abs raised in different species);
and (6) Signals
S obtained with NA probes are expected to be more quantitative than with Abs,
especially
when directly labeled, yet can also be amplified if needed (biotin, etc.).
Using all the sequence information available on targeted bodies (e.g.,
biological
warfare organisms), specific oligonucleotide probes to each of them can be
designed. There
is much less risk of stumbling onto a sequence shared with other organisms
than is the case
with cross-reacting epitopes, because each of the designed probes can be
directly compared
with the entire content of the bacterial/viral nucleic acids databases and
designed to be
unique to a particular target. Fairly short probes (e.g. 20-mers) can be used
to maximize cell
wall/capsid penetration and access to intracellular nucleic acid targets. The
target sequence
unique to a target body can be chosen to be on an abundantly expressed RNAs to
maximize
sensitivity, e.g., sequences in the ribosomal RNAs. For viruses, probes can be
designed that
are selective for the most abundantly expressed genes.
For single labeling experiments, the digoxigenin detection system (Zarda et
al., J.
Gen. Microbiol. 137:2823-2830, 1991) can be used. This system is commercially
available
as a kit from Boehringer Mannheim. In most instances, however, multiple
labeling may be
required, which is not possible with this system. Rather, the oligonucleotides
can be
synthesized in the presence of nucleotides conjugated to a fluorescent dye
(e.g., one from
Genset Corp.). If signal enhancement is required or sought, the
oligonucleotides can be
marked with a tag (e.g. biotin) during synthesis. In this case, each tagged
probe would be
reacted separately with one of several different streptavidin-label complexes,
where the label
is one of, for example, 24 fluorophores. These pre-reacted oligo probes
complexes should be
small enough to diffuse freely through bacterial membranes. If such is not the
case, however,
the cells can be permeabilized with lysozyme/EDTA.
As mentioned above, a wide variety of fluorescent molecules are known and
available. It is estimated that over 50,000 dyes are available from Eastman
Kodak, Polaroid,
Fuji Film, and Molecular Probes (www.probes.com). Examples of molecules
suitable for
nucleated cell targets include DAPI, propidium iodide, acridine orange, and
YOPRO.
23
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
Detection System Components
The various components required for the detection systems are commercially
available. The detector can be any means (e.g., instrument, combination of
mirrors and/or
lenses suitable, photomultiplier, or other detecting means) for measuring,
recording, imaging
or detecting light, fluorescence or other energy transmission, including
excitations,
emissions, and the like. In general, the system includes a fluorescent
microscope with a
motorized stage (e.g., Nikon Microphot-FXA or Nikon Eclipse 1000, both from
Nikon,
Japan; stages from Ludl Electronic Products Ltd., Hawthorne, NY or Axioplan 2
IE MOT
from Zeiss, Germany), fluorescence filters (either included or made to order
from Omega
Optical, Brattleboro, VT), a camera (e.g., CCD 72 camera from DAGE-MIT, Inc.,
Michigan
City, IN; AxioCam from Zeiss, Germany; or SpectraVideo camera from Pixelvision
(www.pixelvision.com)), and a computer having a printer, monitor, storage
medium, display
and software necessary for implementing the invention. Many of the listed
components are
available from vendors such as Nikon, Zeiss, Georgia Instruments (Roswell,
GA), Vaytek
(Fairfield, IA), Applied Imaging, Inc. (www.micrometastasis.orglmetfsl.htm),
and
Chromavision Medical Systems, Inc. (www.chromavision.com).
Whatever components are used, the system should be capable of carrying out the
following steps or variations and equivalents thereof
1) counting the number of target bodies (e.g. cancer cells) per specimen field
(e.g.,
glass microscope slide), subdivided into categories of bodies containing the
second or third fluorophore, or both;
2) saving (e.g., recording, imaging, storing on a data storage medium) an
image of
each target body;
3) storing the x,y coordinates for each target body; and
4) counting the total number of bodies on the slide.
The analysis is performed by scanning the specimen field. Scans can be
performed at all
magnifications provided by the microscope hardware. The user can choose to
scan the
specimen field using any filter set (single, dual, or triple). Scans can be
run independently.
The algorithm for the detection and identification of target bodies is based
on
commercially available software for biological image analysis (e.g. Image Pro
Plus from
24
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
Media Cybernetics, www.mediacy.com; or KS 400 from Kontron, Germany). The
inclusioi
criteria for the detection of target bodies can be for example:
a) fluorescence intensity threshold in the second and third fluorescent
channels;
b) area and shape in the second and third fluorescent channels to distinguish
true
target bodies (e.g. intact cells) from false target bodies (e.g. dirt,
debris); and
c) the signals) of the second and/or third fluorescent channels should always
colocalize with the signal from the first fluorescent channel (e.g. DAPI
signal).
Before each scan, the inclusion criteria for a target body are defined by the
user. After the
scan, a count for all target bodies that fulfill the inclusion criteria (see
above) should be
displayed and subdivided into target bodies that exhibit second, third, or
both fluorescent
labels. All target bodies that fulfill the inclusion criteria are imaged and
stored as 3-color
RGB-image (step 2 above). At the end of the scan, all images are displayed in
form of a
gallery of images with the option of zooming into each image. For all target
bodies that
fulfill the inclusion criteria (see above), the x,y-coordinates are stored and
the user can recal
each position and automatically move the stage to that position (step 3
above). This option
allows the user to recheck every detected target body under high microscope
magnification.
It is also possible to recall the corresponding image that was taken at a
specified position.
During each scan, the total number of cells (based on the first fluorophore,
e.g., DAPI signa
should be counted and displayed at the end of the scan (step 4 above).
User Interphase with Detection S s
1. Setup of the scan. At the beginning of the scan, the user is prompted to
give the
following information and to choose the parameters of the scan:
1. slide identification(s);
2. number of slides to be scanned;
3. magnification of the scan (choose objective); and
4. filter sets) of the scan (choose between single, dual/triple filter, or
alternate filters during the scan).
Based on the given information, an initial image is displayed and the camera
is set up (adju;
brightness and contrast). The user must define the inclusion criteria for the
positive cells an
choose:
1. intensity threshold;
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
2. lower and upper limit for the area; and
3. shape criteria.
2. Scan. After the initial setup, the scan starts automatically and analyzes
the slides)
according to the specifications.
3. Data output azzd storage. For each slide, the following information is
displayed
and saved:
1. number of target bodies;
2. image of each target body and corresponding coordinates on the stage; and
3. total number of target bodies on the slide.
The information 1-3 immediately above is stored in a folder named and defined
by the user
(identification of the slide).
4. Manual confiz~rnatiorz of positive cells. The user can manually select a
stored image
and recall the position were the image was taken. The stage automatically
moves to that
position and the field can be viewed through the eyepieces.
Speed is a fundamental parameter for evaluation of automated rare event
analysis
systems. The system described in Example 1 below takes about one hour to scan
1 million
cells for positive events (e.g. CK positivity) and for the total cell count.
Much faster systems
may be employed, using a more sensitive charge-coupled device (CCD) camera and
a faster
computer. Such a system could bring down the processing time to a few minutes
per million
cells. This flow through rate is comparable to flow cytometry, yet retains the
ability to
observe each positive event at higher magnification or with different optics,
for
morphological confirmation if desired.
Without further elaboration, it is believed that one skilled in the art can,
based on the
above disclosure and the Examples below, utilize the present invention to its
fullest extent.
The following examples are to be construed as merely illustrative of how one
skilled in the
art can practice the invention, and are not limitative of the remainder of the
disclosure in any
way. All references cited herein, whether in print, electronic, computer
readable storage
media or other form, are expressly incorporated by reference in their
entirety, including but
not limited to, abstracts, articles, journals, publications, texts, treatises,
Internet web sites,
databases, patents, and patent publications.
26
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
Example 1: Rare Event Imaging System for Cancer Cells
Materials & Methods
Collection of blood and bone marrow specimens. Five to ten milliliters of
blood or
bone marrow were drawn from control subjects or patients with a diagnosis of
breast or small
cell lung cancer and deposited in Vacutainer tubes containing EDTA as
anticoagulant (Becton
Dickinson, Franklin Lakes, N~. All samples were obtained with informed consent
from the
subject or patient and were processed for microscopic analysis within 24 hours
of collection.
Cell lines. The breast carcinoma cell line MCF-7 and the small cell lung
cancer cell
line SW2 were purchased from American Type Culture Collection (ATCC),
Manassas, VA,
and used to evaluate the staining protocol below and to determine the
sensitivity of the Rare
Event Imaging System. Cell lines were maintained in Dulbecco's modified
Eagle's medium
(MCF-7) or RPMI 1640 (SW2) containing 10% fetal calf serum, 100 U/ml
penicillin, and 0.1
mg/ml streptomycin.
Sample preparation for microscopic analysis. Blood or bone marrow samples were
mixed with 2 volumes of 0.17 M ammonium chloride, incubated at room
temperature (RT)
for 40 minutes, and centrifuged at 800 x g for 10 minutes at RT. The cell
pellet was then
washed and resuspended in phosphate-buffered saline (PBS). The total number of
living
peripheral blood mononuclear cells (PBMC) or nucleated bone marrow cells was
counted
using Trypan blue dye exclusion. The cells were attached to adhesive slides
(Paul Marienfeld
GmbH & Co., KGS Bad Mergentheim, Germany) at 37°C for 40 minutes, and
the slides were
then blocked with cell culture medium at 37°C for 20 minutes. The total
number of cells
applied per slide was about 1.5 x 106. The total adhesive area, divided into
three separate
circles, was about 530 mm2.
For the single labeling of cytokeratin, cells were fixed in ice-cold methanol
for
5 minutes, rinsed in PBS, and incubated with a rabbit anti-cytokeratin
antiserum directed
against class I and II cytokeratins (Biomedical Technologies, Stoughton, MA)
at 37°C for
1 hour. Subsequently, slides were washed in PBS, incubated with rhodamine-
conjugated
anti-rabbit antibody (Jackson Immuno Research, West Grove, PA) at 37°C
for 30 minutes,
counterstained with 0.5 ~.g/ml 4',6-diamidino-2-phenylindole (DAPI; Molecular
Probes,
Eugene, OR) in PBS at RT for 10 minutes, and mounted in glycerol-gelatin
(Sigma, St.
27
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
Louis, MO). Processed slides were stored at RT and analyzed microscopically
within a
month.
For the double labeling of cytokeratin and the cell surface antigens Ep-CAM or
GD2,
the cells were fixed in 1% paraformaldehyde in PBS (pH 7.4) at RT for 5
minutes, washed in
PBS, and blocked with 20% human AB serum (Nabi Diagnostics, Boca Raton, FL) in
PBS at
37°C for 20 minutes. Subsequently, primary antibodies directed against
Ep-CAM
(monoclonal mouse KS1/4 antibody) or GD2 (monoclonal mouse 1418 antibody) were
applied at 37°C for 1 hour. (Both antibodies were kindly provided by
Dr. Kim-Ming Lo,
Lexigen Pharmaceuticals; Lexington, MA.) Antibodies directed against Ep-CAM
are
available from several vendors, e.g., monoclonal mouse anti-human epithelial
specific
antigen is available from Biomeda, Foster City, CA; monoclonal anti-human
epithelial
antigen (Ber-EP4) is available from Accurate Chemical & Scientific Corp.,
Westbury, NY;
and monoclonal HEA-FITC antibody is available from Miltenyi Biotec, Bergisch
Gladbach,
Germany. Antibodies directed against GD2 are available from Matreya, Inc.,
Pleasant Gap,
PA. Cells were then washed, fixed in ice-cold methanol for 5 minutes, blocked
with 20%
human AB serum, and incubated with anti-cytokeratin antiserum at 37°C
for 1 hour.
Secondary antibodies (FITC-conjugated anti-mouse and rhodamine-conjugated anti-
rabbit
antibodies; Jackson Immuno Research) were mixed and applied at 37°C for
30 minutes.
Nuclei were counterstained with 0.5 pg/ml DAPI in PBS. Doubly labeled cells
were
mounted in Gel/Mount (Biomeda, Foster City, CA). Slides were stored at
40°C and analyzed
microscopically within a week.
Tunaor cell dilutions for determination of sensitivity. To determine the
sensitivity of
the detection for cytokeratin-positive (CK+) cells, MCF-7 breast cancer cells
were serially
diluted in PBMC of a healthy blood donor. The dilutions tested were 1:103,
1:104, 1:105, 1:2
x 105, 1:5 x 105, and 1:106. Solutions were attached to adhesive slides and
processed for
cytokeratin labeling as described above. Up to 8 adhesive slides were prepared
and scanned
per dilution. Samples were analyzed for the number of tumor cells per slide
and related to
the total cell count.
Automated naicroscopic detection of tumor cells a~°rd total cell count.
Slides were
automatically scanned using an imaging system, such as for example, a Rare
Event Imaging
System, developed by Georgia Instruments, Inc. (Roswell, GA). The system
employs
28
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
proprietary image processing algorithms to detect rare fluorescent events and
determine the
total number of cells analyzed. It is comprised of an advanced computer-
controlled
microscope (Nikon Microphot-FXA, Nikon, Japan) with autofocus, motorized X, Y,
and Z
axis control, motorized filter selection, and electronic shuttering. Images
were taken by an
integrating, cooled CCD detector and processed in a 60 MHz Pentium imaging
workstation.
In a first step, the slide was automatically scanned for the detection of
positive events
(e.g., CK+ cells) using the rhodamine filter set. The identification of
positive events was
based on fluorescence intensity and area. The (x,y) coordinates of each
positive event were
stored in computer memory, and the image was archived. In a second step, the
slide was
scanned for the total number of DAPI-labeled nuclei per slide, representing
the total cell
count. The total scanned area per slide was 448 mma (84% of the adhesive area)
to avoid
edge effects. At the end of the two scans, the number of positive events and
the total cell
count were given, and a gallery of images containing all positive events was
displayed. The
user could review the images and recall any of the events for further
examination, using the
stored coordinates attached to each image. The field of interest could then be
visualized
using higher magnification and additional filter sets (e.g. fluorescein, or W
filter). Images
of different fluorescent colors could be electronically overlaid for positive
confirmation of
the event and for phenotypic evaluation (multiple labeling). The total
scanning time (two
scans) for one slide was about 1 hour. The two scans could be run
independently, offering
the option of just screening for positive events and thus shortening the
scanning time to 30
minutes per slide.
Results
Evaluation of the cell deposition procedure. One of the most critical steps
during
sample preparation is deposition of the cells onto slides. A qualitative
microscopic
comparison of cell preparations attached to poly-L-lysine/PBS-coated slides
(0.1%; Sigma,
St. Louis), SectionLock Slides (Polysciences, Inc., Warrington, PA}, and
adhesive slides
(Paul Marienfeld GmbH & Co., KG) revealed that the most homogeneous cell
monolayers
(optimal cell density with minimal overlap) was obtained with the slides from
Paul
Marienfeld. The slides contain a charged surface for the attachment of living
cells. To
further validate our deposition technique for different types of samples, the
total number of
cells as determined by the Rare Event Imaging System was compared with the
number of
29
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
cells originally deposited onto the slides. With optimization, any adhesive
surface (e.g.,
coated with a positively charged substance such as poly-L-lysine) can be used.
Table 1
shows a high cell recovery (89%) for peripheral blood of healthy blood donors,
but a
somewhat higher cell loss in samples from cancer patients (64, 58, and 73%
recovery for PB,
BM and SC samples, respectively; p < 0.05 for PB and BM vs Normal PB, by t-
test).
Table 1
Sample type Cell count/slide Range (n) Recovery
Normal PB 1,120,237 ~ 93,372733,833 - 1,470,633(8) 89%
Cancer PB 811,400 ~ 89,039*223,393 - 1,473,777(17) 64%
Cancer BM 731,945 ~ 72,906*157,110 - 1,459,414(25) 58%
Cancer SC 915,983 ~ 95,806 76,745 - 1,631,660(23) 73%
Peripheral blood (PB), bone marrow (BM), or peripheral blood stem cell (SC)
samples from
healthy subjects (Normal) or cancer patients were prepared as described in
"Materials and
Methods," and 1.5 x 106 cells were applied to each adhesive microscope slide.
Cells were
counted (based on DAPI labeling) on the number of slides indicated for each
group (n), and
results are expressed as mean ~ SEM. For the calculation of recovery, note
that the area
scanned on each slide is 84% of the total adhesive area (see "Materials &
Methods").
Asterisks mean that p < 0.05 vs Normal PB by t-test.
Se~rsitivity of the detection method To explore the sensitivity of the Rare
Event
Imaging System, PBMC samples that had been spiked with breast cancer cells
(MCF-7) were
prepared and processed for cytokeratin labeling. The brightly stained
epithelial MCF-7 cells
could easily be distinguished from the mesenchymal background of the white
blood cells.
The sensitivity of detection of CK+ cells was tested with increasing tumor
cell dilutions
(MCF-7/PBMC) as described in "Materials 8~ Methods." Cancer cells in expected
quantities
could be detected up to the most diluted samples tested, 1 MCF-7 cell per 106
PBMC
(Table 2; expected and observed curves not statistically different, x2 test).
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
Table 2
Cells added Total number Total cellCells detected
of
per 106 PBMCcells detected count per 106 PBMC
1000 1789 1.94 x 922
106
100 169 1.79 x 95
106
27 2.3 5 x 12
106
5 38 5.16 x 7
106
2 11 3.94 x 3
106
1 ' 13 6.13x106 2
Double-labeling of tumor cells. In order to increase the specificity of rare
event
detection and to further characterize the cancer cells identified, a staining
protocol that allows
the detection of intracellular cytokeratin and a cancer cell surface marker
simultaneously was
developed. The double-labeling procedure consists of two sequential steps:
first fixing the
cell surface and labeling for Ep-CAM or GD2, and second permeabilizing the
cells and
staining for intracellular cytokeratin. The double-labeling protocol was
optimized in the
cancer cell lines MCF-7 (breast cancer) and SW2 (small cell lung cancer).
Fluorescence
10 microscopy indicated that SW2 cells were ei~iciently labeled with anti-GD2
antibody and
anti-cytokeratin antiserum. The sequential fixation preserved the antigenic
sites of both
proteins with, regard to their cellular localization, as demonstrated in the
optical sections
taken with a confocal laser scanning microscope. The stained cells clearly
showed
cytokeratin in the cytoplasm (red) and GD2 at the cell surface (green). The
expression levels
of both proteins was quite heterogeneous within the cell population. A similar
result was
obtained when MCF-7 cells were doubly labeled with the anti-Ep-CAM antibody
and the
anti-cytokeratin antiserum. Control experiments in which one of the primary
antibodies was
omitted but both secondary antibodies were applied revealed no cross-
reactivity between the
two detection systems.
To further validate the staining protocol, PBMC that had been spiked with MCF-
7 or
SW 2 cells were labeled. The goal was to obtain a bright fluorescent signal of
the cancer
cells and a low background signal from the surrounding PBMC. The two most
important
factors for achieving this goal were found to be the sequential application of
the primary
antibodies and two blocking steps (20% human AB serum in PBS) prior to the
incubation
with the primary antibodies. Fluorescence microsocpy indicated that the doubly
labeled
MCF-7 cells could clearly be distinguished from the surrounding PBMC. At
higher
magnification, the intracellular cytokeratin labeling and the surface staining
of Ep-CAM was
31
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
confirmed. Similar results were obtained with PBMC spiked with SW 2 cells and
doubly
labeled for GD2 and cytokeratin.
The double-labeling protocol was also applied to peripheral blood and bone
marrow
samples from cancer patients. In an example of a GD2/cytokeratin-positive cell
from
peripheral blood of a patient with small cell lung cancer, fluorescence
microscopy showed an
Ep-CAM/cytokeratin-positive cell from bone marrow of a breast cancer patient.
In this
example, the cancer cell was not only bigger than the surrounding bone marrow
cells but it
also exhibits the distinct localization of the individual stains: cytokeratin
(red) in the
cytoplasm and Ep-CAM (green) concentrated towards the cell periphery at the
cell
membrane.
Detection of cytokeratizz positive azzd doubly positive cells izz normal blood
samples.
To evaluate the specificity of the single- and double-staining protocols,
blood samples from
healthy donors were analyzed. The number of "positive" cells was compared
among
methods using the single cytokeratin or double cytokeratin/Ep-CAM or
cytokeratin/GD2
labeling methods. Fluorescence microscopy indicated that 16-18% of the PB
samples scored
positive for cytokeratin using any of the protocols, with the number of CK+
cells ranging
from 1 to 26 labeled cells per 106 white blood cells. In contrast, when the
samples were
processed with the double-labeling protocol, positivity was almost completely
eliminated
from samples of healthy subjects (a single doubly positive cell was observed
in a total of 77
PB samples).
Evaluation of spatial azzd temporal variations ih sample collection. To assess
a
possible heterogeneity in the distribution of CK+ cells in different areas of
the bone marrow,
paired BM samples from the right and the left iliac crests of the same patient
were obtained
and analyzed. Out of 24 pairs, 21 showed concordant results (Fisher exact
test) with regard
to cytokeratin positivity (Table 3A). The occurrence of CK+ cells in
peripheral blood
samples temporal fluctuations was also tested. Two SC samples from each of 96
patient were
taken at consecutive days but without therapeutic intervention. Paired samples
showed a
statistically significant concordance with regard to cytokeratin positivity
(Table 3B).
32
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
Table 3 A
BM 2
+ -
BM1 + 11 1
- 2 10
Table 3B
SC 2
+ -
SC 1 + 19 9
- 8 60
Detection of cytokeratin positive cells ih cancer patient blood aid bone
marrow
samples. To demonstrate the power of the Rare Event Imaging System, 355
peripheral blood,
bone marrow, and stem cell samples were analyzed. These samples were obtained
from
breast cancer patients before autologous bone marrow transplantation but after
high-dose
chemotherapy. The samples were screened using the single cytokeratin labeling
method. In.
an example of two CK+ cells from peripheral blood of a breast cancer patient,
the positive
cells showed clear cytoplasmic labeling whereas the surrounding blood cells
were not
stained. CK+ cells were found in 52% of the bone marrow, 34% of the peripheral
blood, and
27% of the stem cell samples (Table 4).
33
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
Table 4
Total CK+ samples CK+ samples
samples (All) (z 9 CK+/106 PBMC)
Count % Count
BM samples 63 33 52 25 40
stages 11/III20 7 35 5 25
stage IV 43 26 60 20 46
PB samples 59 20 34 14 24
stages )I1I1113 2 1 S 2 1
S
stage IV 46 18 39 12 26
SC samples 233 64 27 29 12
stages 11/III49 11 22 4 8
stage IV 184 53 29 25 14
For Table 4, bone marrow (BM), peripheral blood (PB), and peripheral blood
stem cell (SC)
samples from a total of 156 patients were analyzed for cytokeratin-positive
cells and total cell
count. Note that there were multiple samples from some patients whereas for
others, only
one kind of sample could be analyzed. "CK+ samples (All)" refers to the number
of samples
with at least 1 CK+ cell. CK+ samples (>_ 9 CK+1106 PBMC) refers to number of
samples
with 9 or more CK+ cells per 106 PBMC (mean + 2 SD of CK+ cells in Normal PB;
Table S).
The highest numbers of CK+ cells per sample were 504/106 for BM, 371/106 for
PB, and
1020/106 for SC.
Table 5
CK+ labeled Doubly labeled
Markers) Total positive CK+ /106 CK+ /106 Positive DBL+ /1C
samples samples (all samples) (CK+ samples (DBL+
CK 57 10 (17%) 1.18 + 0.53 7.28 ~ 2.59 --- ---
CK/Ep-CAM 43 7 (16%) 0.46 + 0.21 2.85 ~ 0.81 1 (2.3%) 1.4
CK/GD2 34 6 (18%) 0.78 + 0.44 4.41 + 1.98 0 (0.0%) 0
For Table 5, blood samples from healthy blood donors were labeled for
cytokeratin alone, or
doubly labeled for CK/Ep-CAM or CK/GD2 (see "Material and Methods"). Positive
samples
1 S were those containing CK+ cells (in single-labeling) or doubly labeled
cells (in double-
labeling). Numbers of positive cells in each category are expressed per 106
cells analyzed,
34
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
and are given as mean + SEM (except for the single positive cell in one sample
containing
7.14 x 105 cells, in the CK/Ep-CAM group). DBL+ means doubly labeled
As seen in Table 4, the frequency of CK+ cells in the positive samples varied
from
11106 to 1020/106: However, many PB samples from normal subjects displayed a
small
number of CK+ cells, and these were found to be false positive cells, based on
the double-
labeling experiments (Table 5). Therefore, to declare definite positivity in
PB samples from
cancer patients, a cut-off point was set at the mean number of CK+ cells plus
2 times the
standard deviation as observed in the control samples, i.e., 9/106. Applying
this threshold, a
higher degree of cytokeratin positivity in bone marrow (40%) compared to
peripheral blood
(24%) or stem cell preparations (12%) was still found (Table 4). Furthermore,
patients with
stage IV disease were found to be cytokeratin-positive in a significantly
higher percentage
than patients with stages II/TTT disease, in all types of samples analyzed
(Table 4).
In summary, an automated analysis system for the detection of cells of
interest that
occur at low frequencies (rare events) was developed using dual- or multiple-
marker
analysis. The preparation procedure for the microscopic analysis of blood or
bone marrow
samples was optimized for automation and included lysis of red blood cells,
deposition of
mononuclear cells onto adhesive sides, and immunofluorescent labeling of the
sample.
Slides were then examined at low magnification under a fluorescence microscope
fitted with
a motorized stage, and all the fluorescent events are imaged and catalogued in
a computer
database for later retrieval. For automated image analysis it is crucial to
work with
secondary antibodies that give a bright signal while maintaining a low
background. We have
recently tested dyes of the Alexa-series (Molecular Probes) that give very
bright and stable
fluorescence signals. Fluorescently labeled slides should be analyzed within
one week. If
longer storage is desired, a mounting medium that maintains stable fluorescent
signals should
be used. We found that the use of the Prolong Antifade Kit (Molecular Probes)
gave
excellent results after 3 month storage of the slides at 40°C.
Example 2: Optimization of Rare Event Ima ig~n~S s
Adaptation and optimization of basic procedures for sample preparation cell
attachment and
staining
The slides used(Paul Marienfeld GmbH & Co. KG, Bad Mergentheim, Germany)
contain 3 adhesive circles of 150 mm2 each, onto which the cells are seeded.
The adhesion
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
procedure developed for human cells is adapted to the processing of
microorganisms.
Selected parameters tested include the time of contact with the adhesive
slide, the
temperature, the pH, the composition of the buffer and its ionic strength.
Separate tests are
performed with a bacteria (e.g., E. coli, B. subtilis, h clrole~ae) and
viruses (e.g. reovirions)
to verify for possible variability in their characteristics of adhesion to the
slides. Detection is
performed using DAPI or acridine orange (which labels RNA for RNA viruses).
Since an
even cell monolayer is essential for automation, testing with other cell
deposition systems
(e.g. cytospin using a Shandon Cytocentrifuge; Cytotek Monoprep from Sakura,
Torrance,
CA; and ThinPrep from Cysyc, Boxborough, MA) is used for comparison purposes.
Develop computer software for REIS automation
We have automated the whole sequence of steps for our original analyzer
(Kraeft et
al., Clin. Cancer Res. 6:434-442, 2000). As necessary, an appropriate camera
driver is
obtained for the particular system to be implemented. The writing of such
drivers is well
within routine skill in the art of computer programming. The REIS employs
image
processing algorithms to detect rare fluorescence events. Images are taken by
the detector
and processed in a PC-based imaging workstation. The software performs the
detection of
fluorescent signals (antigen-positive organisms) as well as total cell count
(e.g., based on
DAPI/acridine orange staining), automated signal positioning, image archiving,
and image
processing. Initially, this is be done with one or two fluorophores (e.g.
AlexaFluor488 and
AlexaFluor568). The multiplex detection system can be expanded to accommodate
multiple
dyes (e.g., up to 24 dyes), and the software superimposes each fluorescent
signal observed
with each dye, with the corresponding image obtained with, e.g., DAPI/acridine
orange stain.
Improvement to the multiple labeling system
The methods herein are useful not only to monitor the presence of bacteria and
viruses in air or water samples, but also to detect and ident~ pathogens, in
particular those
identified as BW agents. These axe numerous, and although it would be possible
to generate
as many slides as there are agents to be tested for, this would be
impractical. Rather, one
aspect involves development of a multiplex system whereby various fluorophores
are be
used, whose excitation/emission spectra can be differentiated.
As discussed above, an estimated 50,000 fluorescent dyes are available. Thus
it is
possible to screen this collection for a set of at least 24 dyes that give the
brightest
36
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
fluorescent signals (for maximum sensitivity) whose excitation and emission
spectra can be
differentiated, and that can be conveniently conjugated to antibodies, via an
isothiocyanate
bridge. A set of dyes with similar fluorescence intensity would also be
favorable.
A set of filters to match and discriminate at least the emission peaks of the
dyes
chosen, plus DAPI and acridine orange, are selected for use in the methods.
The excitation
wavelength is controlled either by a separate set of filters or by using a
narrow-band prism
for the incident light. The wheels carrying the fluorescence filters are
modified to
accommodate all the excitation and emission filter combinations required for
the
discrimination of the distinct fluorophores.
Example 3' Development of Methods for Pathogen Detection by
ImmunocytochemistrY
Generation of fluorescently labeled antibodies. The first requirement for
immunocytochemical assays is the generation of good antibodies. When available
commercially or otherwise, existing antibodies directed against surface
antigens of BW
agents are selected for use. In other cases, irradiated (killed) samples of
the organisms of
interest (from the CDC, USAMRIID, etc.) are selected for use and the
production of
monoclonal antibodies (mAbs) to exposed epitopes is performed; If any of these
organisms
carry common surface epitope that would cause cross reaction, or if reliably
"killed"
organisms cannot be obtained, one or several antigens specific to the species
can be selected,
and either purified, expressed from their cloned genes, or mimicked by a
chemically
synthesized peptide, and used as immunogens. All antibodies are either
directly conjugated
with fluorescent molecules or used in combination with secondary fluorescently
labeled
antibodies. Testing of directly labeled antibodies is performed by FACS
analysis for
specificity against other phylogenetically related species, especially those
described herein.
Antibodies for a variety of bacteria, rickettsiae, viruses and fungi listed
above or to
other suitable model microorganisms can be used to develop a pathogen
detection rare event
imaging system (REIS). Each of six different antibodies is conjugated with 4
different dyes,
for a total of 24 distinguishable fluorescently labeled antibodies.
Ultimately, a multiplex
detection system with 24 distinguishable fluorophores (conjugated to a set of
24 specific
antibodies), would allow one to monitor and positively identify all the known
or suspected
BW agents directed to humans on only 2 slides (i.e. 2 sets of 24 antibodies),
and nearly all
the BW agents listed herein on only 3 slides.
37
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
Immu~ocytochemical detection ofpathogens immobilized orato slides. Samples of
attenuated strains or irradiated (killed) organisms for the 6 species to which
antibodies are
raised, (if pathogenic, from the CDC, USAF, etc.), are applied in known
numbers
directly onto adhesive glass slides for analysis using the REIS.
In a first set of experiments, known numbers of organisms from each species
individually, are examined to estimate the level of sensitivity of their
cognate labeled
antibodies, using the REIS; each should be able to detect single, individual
organisms, and
the enumeration should be quantitative. DAPI is used to stain DNA as before,
or acridine
orange for RNA viruses. All parameters of the procedure (temperature, buffer
composition,
antibody concentration, etc.) are optimized for each organismlantibody set,
with a special
attention to the minimization of the time of incubation. Initial conditions
are essentially as
described in Example 1.
In a second set of experiments, every pair of the organisms of interest,
seeded on the
slides in proportions of 10%-90% and 90%-10%, are examined to ascertain the
absence of
cross reactivity in the context of REIS analysis.
A third set of experiments test the multiplex set-up; using a mixture of 6
organisms
(bacteria/rickettsiae, viruses, and mixture of the two), in the proportions of
5, 10, 15, 19, 23
and 28%, allowing for a verification of the detection efficiency using
multiplex. Then, 24-
strong multiplex experiments are performed using the 4 preparations of each of
the 6
antibodies, with the organisms seeded in the same proportions as above.
Throughout these experiments, data is collected on detection efficiency. This
can be
performed on 75 organisms described herein (or any other BW agent). Such
information is
useful to determine sets of organisms that can be analyzed together
efficiently, a point that
may be especially important if, for example, the requirements for adhesion
onto slides (see
Example 2) or for the incubation with the antibodies varies between species.
ImmutZOCytochemical detection of pathogens in environmental samples or human
body fluids. As discussed above, optimized preparation procedures for the
immunocytochemical detection of microorganisms are applied to environmental
(air and
water) and human (blood and other body fluids) samples. Whereas waterborne
pathogens
can be processed directly from the source, airborne bacteria and viruses
require a special
sampling procedure to immobilize them onto the slides. Several suitable air
sampling
38
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
devices exist on the market, including the BioSampler from SKC. This is a
vacuum-drive
all-glass impinger device that uses air nozzles tangential to the surface of
the collection fluff
rather than bubbling air in the fluid, minimizing particle bounce and reducing
re-
aerosolization. When operated at an air flow rate of 12.5 L/min with water or
a liquid of
similar viscosity as the collection fluid, the collection efficiency of the
BioSampler~ is clos
to 100% for particles as little as 1 ~.m in diameter, and still approximately
90% at 0.5 ~m a:
80% at 0.3 Vim. As such, the BioSampler~ is an excellent device for the
collection of
airborne bacteria, fungi, pollen, and viruses: most bacteria are between 1 and
10 ~.m in
diameter, and many viruses have a size in the lower end of this range (e.g.
Ebola virus, 100
x 80 nm). Other air samplers are also suitable. For example, an alternative
from SKC whit
may be convenient for certain sample types is the Air-O-Cell sampling
cassette, in which t1
airborne particles are accelerated and made to collide with a tacky slide
which is directly
suitable for various staining procedures and microscopic examination. However,
this
collection method is inefficient for particles smaller than 2 or 3 Vim.
The main parameters to be modified in environmental sampling are the time of
sampling, and the collection fluid composition. Various fluids can be tested
and compared
direct inoculation tests with known amounts of organisms, for their capacity
to support
adhesion to the slides.
The analysis of human body fluids is exemplified by the analysis of blood
samples.
First, normal blood from donors it is spiked with a known amount of pathogen
(bacteria an.
viruses). We will analyze the recovery of the microorganism, establish a
detection thresho:
and compare manual with automated analysis. Second positive blood cultures
from
microbiological laboratories (for example, the Dana-Farber Cancer Institute)
are obtained
and utilized in the detection method for bacteria described herein and
compared with the
clinical result (based on bacterial culture). Additionally, negative blood
samples from
patients can serve as controls.
Development of methods for pathogen detection by izz situ hybridization. Even
though immunofluorescence (IF) is currently the method of choice for Rare
Event analysis.
there are several reasons why detection using nucleic acid probes and an in
situ hybridizati~
(ISH) approach may be preferable. These are summarized in Table 7. Thus, such
approaches offer useful alternatives to the use of antibodies in IF.
39
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
_ Table 7: Possible advantages of ISH over 1F procedures
1. Nucleic acid (NA) probes are a lot easier, quicker, and cheaper to generate
than antibodies
(Abs).
2. NA probes can be grown at will and inexpensively (monoclonal Abs too, but
not
polyclonal).
3. NA probes are expected to be more consistent than Abs (especially
polyclonal; can even
choose probes with matching Tm, for multiple labeling (multiplex)
experiments).
4. NA probe hybridization to its cognate RNA target can be better controlled
than antibody
interaction with its epitope (hybridization temperature, ionic strength,
etc.).
5. Multiple-label experiments are much simpler with NA probes (simply
incorporate a
nucleotide conjugated to different labels, or biotin and then various
streptavidin-label
complexes; in IF, labeling of primary Ab may interfere with its binding, and
when a 2"a
Ab is used for detection, IF requires the use of primary Abs raised in
different species).
6. Signals obtained with NA probes are expected to be more quantitative than
with Abs,
especially when directly labeled, yet can also be amplified if needed (biotin,
etc.).
Generation of nucleic acid probes. Using all the sequence information
available on
targeted organisms, we can design specific oligonucleotide probes to each of
them. There is
S much less risk of stumbling onto a sequence shared with other organisms than
was the case
with cross-reacting epitopes (see Example 2) because each of the designed
probes can be
directly compared with the entire content of the bacterial/viral nucleic acids
databases. Use
of fairly short probes (e.g. 20-mers) can maximize cell wall/capsid
penetration and access to
intracellular nucleic acid targets, and abundantly expressed RNAs can be used
to maximize
sensitivity. In this instance, selection of sequences in the ribosomal RNAs to
the cellular
organisms of interest that are specific to each species is useful. For
viruses, probes are
designed to the most abundantly expressed gene.
For single labeling experiments, we can use the digoxigenin detection system,
which
is commercially available as a kit (Boehringer Mannheim). In most instances,
however,
multiple labeling may be required, which is not possible in this system.
Rather, the
oligonucleotides will be synthesized in the presence of nucleotides conjugated
to the
fluorescent dye (Genset Corp.). If signal enhancement is required or sought,
we may mark
the oligonucleotides with a tag (e.g. biotin) during synthesis. In this case,
each tagged probe
would be reacted separately with one of several different streptavidin-label
complexes, where
the label is one ofthe 24 fluorophores from above. These pre-reacted oligo
probes
complexes should still be small enough to diffuse freely through bacterial
membranes. If
such is not the case, one can permeabilize the cells with lysozyme/EDTA.
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
Detection of pathogens i»amobilized onto slides by in situ hybridization.
These
experiments will parallel those described in Example 2 above. The "first set
of experiments"
requires only single labels and is performed with the digoxigenin system. For
all others,
fluorescently labeled oligonucleotides or the biotinylated oligo +
streptavidin-label complex
detection method described above are used. Again, data collection on detection
effciency
throughout these experiments can be evaluated to determine the optimal sets of
BW agents
(up to 24) that can be monitored together, as a multiplex assay on a single
slide.
Detection of pathogens in environmental samples and human body fluids by in
situ
hybridization. These experiments parallel those described in Example 2.
Environmental and
human samples are analyzed in parallel, using immunocytochemistry and in situ
hybridization.
Example 4' Single Label Protocol
All antibodies are diluted in PBS containing 20% human AB serum.
1. Lyse blood: 11m1 isotonic NH4C1 for 3m1 blood in a 15m1 conical tube. Leave
at room
temperature for 40 minutes.
2. Centrifuge at 1500rpm for 5 minutes.
3. Remove supernatant of NII4C1 and erythrocytes, leaving a pellet of white
blood cells.
Resuspend in the pellet in lOml PBS.
4. Centrifuge at 1500rpm for 5 minutes.
5. Remove supernatant; resuspend pellet in 1, 0.5, or 0.25m1, depending on the
size of the
pellet.
6. Make a dilution of cells, trypan blue and PBS for cell counting on the
haemocytometer.
1:100 DILUTION: 10u1 trypan blue, 10u1 cells, 980u1 PBS
7. Calculate and adjust to have 5x10~5 cells in 100u1 per circle (on adhesion
slide)
8. Plate cells on adhesive slides; allow 40 minutes for cell attachment at
37°C.
9. Add 60u1 of 50:50 media per circle to the slides; incubate at 37°C
for 20 minutes.
10. Put slides into 2% paraformaldehyde for 20 minutes, room temperature.
11. Rinse slides 2X 3 minutes.
12. Put slides in -20°C methanol for 5 minutes.
13. Rinse in PBS 2X 3 minutes.
14. Add 60u1 of 20% human AB serum to each circle for 20 minutes at
37°C. DO NOT
RINSE! Tap off.
15. Add 60u1 of primary antibody (e.g. anti-cytokeratin) to each circle.
Incubate at 37°C for
3 5 1 hour.
16. Rinse in PBS 2X 3 minutes.
17. Add 60u1 of secondary antibody (e.g.anti-rabbit rhodamine). Incubate for
30 minutes,
37°C.
18. Rinse in PBS 2X 3 minutes.
41
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
19. Add 60u1 of DAPI to each circle. Incubate at room temperature for 10
minutes.
20. Rinse in PBS 1X.
21. Place in dH20.
22. Mount with Glycerol gelatin. Put 35u1 on each circle. Put on coverslip.
23. Slides may be stored at room temperature, covered with foil.
Example 5: Double Label Protocol
All antibodies are diluted in PBS containing 20% human AB serum.
1. Lyse blood: l lml isotonic NH4C1 for 3m1 blood in a 15m1 conical tube.
Leave at room
temperature for 40 minutes.
2. Centrifuge at 1500rpm for 5 minutes.
3. Remove supernatant of NH4C1 and erythrocytes, leaving a pellet of white
blood cells.
Resuspend in the pellet in lOml PBS.
4. Centrifuge at 1500rpm for 5 minutes.
5. Remove supernatant; resuspend pellet in 1, 0.5, or 0.25mL, depending on the
size of the
pellet.
6. Make a dilution of cells, trypan blue and PBS for cell counting on the
haemocytometer.
1:100 DILITTION: 10u1 trypan blue, 10u1 cells, 980u1 PBS
7. Calculate and adjust to have 5x10~5 cells in 100u1 per circle (on adhesion
slide)
8. Plate cells on adhesive slides; allow 40 minutes for cell attachment at
37°C.
9. Add 60u1 of 50:50 media per circle to the slides; incubate at 37°C
for 20 minutes.
10. Place slides in 2% paraformaldehyde in PBS at room temperature for 20
minutes.
11. Rinse in PBS 2 X 3 minutes.
12. Add 60u1 of 20% human AB serum to each circle; leave on for 20 minutes,
37°C. Tap
off. DO NOT RINSE!
13. Add 60zi1 of primary antibody (e.g. anti-GD2, anti-GD3, anti-Her-2neu or
anti-
KSA/EpCAM) to each circle. Incubate at 37°C for 1 hour.
14. Rinse in PBS 2X 3 minutes.
15. Add 60u1 of secondary antibody (e.g. anti-mouse Alexa Fluor488). Incubate
for 30
minutes, 37°C.
16. Rinse in PBS 2X 3 minutes.
17. Put slides in -20°C methanol for 5 minutes.
18. Rinse in PBS 2X 3 minutes.
19. Add 60uL of 20% human AB serum to each circle; leave on for 20 minutes,
37°C. Tap
off DO NOT RINSE!
20. Add 60u1 of primary antibody (anti-cytokeratin) to each circle. Incubate
at 37°C for 1
hour.
21. Rinse in PBS 2X 3 minutes.
22. Add 60u1 of secondary antibody (e.g. anti-rabbit rhodamine). Incubate for
30 minutes,
37°C.
23 . Rinse in PB S 2X 3 minutes.
24. Add 60u1 of DAPI to each circle. Incubate at room temperature for 10
minutes.
25. Rinse in PB S 1 X.
26. Rinse in dH20. Let slides dry.
42
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
27. Mount slides with Pro-Long Anti-fade mounting media (Molecular Probes),
coverslip
and seal with clear nail polish (optional).
28. Store at 4°C, covered with aluminum foil.
Example 6' Bacterial Sample Preparation
1. Grow bacteria to semi-log phase ( OD @600nm = 0.5)
2. Spin O.Sml.Bacteria Suspension ( Smin @ SK rpm)
3. Resuspend Bacteria in lml of 10% PBS.
4. Add SOuI of resuspended bacteria to well on slide ( if use Clear-Cell or Ad-
Cell slides
do not require gelatin coating otherwise pretreat slides with gelatin to
improve binding of
bacteria to slide).
5. Dry bacterial suspension 37 Celsius for 30min.
6. Rinse in PBS RT then place in 4% Paraformaldehyde soln for 25-30min.
7. Rinse in PBS RT then drop 100u1 of Triton/well leave for Smin
8. Rinse in PBS RT.
9. Add PreHyb soln 30min @ 46 Celsius ( Prehyb soln; SOng/ul BET42a in Hyb
soln,
Hyb soln see below)
10. *.Hyb for 120min @46 Celsius. Hyb soln with Flourophore conc Sng/ul and
BET42a
conc SOng/ul
11. *Wash for 30 min @ 48 Celsius. Wash soln must be preheated to 48 Celsius.
Run
some Wash soln over slides before soaking in Wash soln for 30 min.
12. *Rinse slides RT PBS
13. *Apply DAPI RT for 5 min.
14. *Rinse DAPI offwith RT PBS, AIR dry then Gel mount, cover slip and nail
varnish
the edges.
* steps from 10 on should be carried in DARK as fluorophore will photobleach.
Hvb Soln
40% Formaldehyde soln
SM NaCI 720u1
1M Tris-HCl (pH7) 80u1
10% SDS 4u1
ddH20 1600u1
Formaldehyde 1600u1
Add Fluorophore to get conc Sng/ul
43
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
Wash Soln
SM NaCI 2.25m1
0.1% SDS O.lml
1M Tris-HCl (pH7.6) 2.0m1
ddH20 95.65m1
FITC, Alexa Fluor 488, Cy 5, Cy3, and Bet42a-blocking labeled oligonucleotides
were
examined. Experiments were performed to establish the specificity of the
probes and signal
intensity of the label. All of the above listed probe sequences reacted
specifically with their
targets but not with the other bacteria.
Example 7: Patient Sampling
A male white patient diagnosed as having a malignant carcinoid tumor of the
midgut
had undergone treatment (don't know). Upon completing the standard treatment
regimen,
the patient was diagnosed as "cancer-free" based upon results of standard
laboratory tests.
The patient's blood was then subjected to the method essentially as described
herein wherein
the sample indicated a white blood cell count of 42 x 106. A sample of 15 x
106 of these
white blood cells were plated and stained using anti-GD2 (14118) and Alexa
Fluor 488
antibodies as primary and secondary antibodies, respectively, followed by anti-
cytokeratin
and anti-rabbit rhodamine as primary and secondary antibodies, respectively,
in the double
label protocol as exemplified in Example 5. The patient's sample showed three
positive cell;
detected by the cytokeratin antibodies. This result indicates that the patient
was, in fact, not
cancer-free. This detection resulted in the patient undergoing a further
treatment regimen to
attempt to more fully eradicate the cancerous cells in a timeframe such that
the prognosis foi
an improved outcome is greater.
Other Embodiments
It is to be understood that while the invention has been described in
conjunction with
the detailed description thereof, the foregoing description is intended to
illustrate and not
44
CA 02436448 2003-07-28
WO 02/062201 PCT/US02/02832
limit the scope of the invention, which is defined by the appended claims.
Other aspects,
advantages, and modifications are within the scope of this invention.
For example, although the procedures described in Example 1 are impressive,
improvements of this "first generation" rare event imaging system can made.
The limiting
factor in some known cell processing rates is mechanical, as several million
cells must be
scanned at fairly high magnification (usually 10-20x). Indeed, system speed is
a problem: at
a processing rate of 1000 cells per minute, it would take about 3.5 days of
non-stop data
acquisition to scan 5 x 106 cells. While the cell density on the slides might
be increased
somewhat, the size and sensitivity of the camera currently in use limit the
magnification to
lOx or Sx at best, which is insufficient and in all practicality precludes
such slow systems
from use in the detection of rare events when time is a factor. While
considerably more rapid
(up to 107 cells in about 4 hours), "first generation" systems also need
improvement for true
usefulness in a clinical (or environmental monitoring) setting.
To address this shortcoming, a "second generation" REIS system can be
employed,
with a goal to shorten the microscope analyzing time from 4 hours to less than
10 minutes.
Based on the technologies available in the rapidly growing electronic imaging
and software
industries, this goal is reasonable. The key is to use a very large field,
extremely sensitive
camera, which would allow the capture of large microscope fields without
scanning the slide.
The idea of using a high-gain digital camera to shorten the processing time of
a "first
generation" system came from the success of Hubble telescope. Far away stars
can be
captured by advanced digital (as opposed to analog) electronic cameras down to
a single
pixel. The size of the grade 1, high quantum e~ciency, back-illuminated charge-
coupled
device ("CCD") chip in some state-of the art cameras is 24.5 x 24.5 mm, with a
pixel array of
1024 x 1024. New CCD chips with a 1600 x 1600 pixel array are also available,
which will
allow one to survey even larger microscopic fields. Utilizing such technology,
it is
envisioned that the image of an entire slide could comprise a chip, and
ultimately a cell
imaged as a single pixel in a large specimen field (e.g., 1 x 109 cells).
45
